A paper copy of the Sequence Listing and a computer readable form of the sequence listing (xe2x80x9cCRFxe2x80x9d) on diskette, containing the file named xe2x80x9c16516.122. txtxe2x80x9d, which is 14,451 bytes in size (measured in MS-DOS), and which was created on Jan. 22, 2002, are herein incorporated by reference.
The invention relates to genetic modification of plants, plant cells and seeds, particularly altering carotenoid biosynthesis, tocopherol biosynthesis, and fatty acid composition.
Carotenoids are pigments with a variety of applications. They are yellow-orange-red lipids which are present in green plants, some molds, yeast and bacteria. Carotenoid hydrocarbons are referred to as carotenes, whereas oxygenated derivatives are referred to as xanthophylls. The carotenoids are part of the larger isoprenoid biosynthesis pathway, which, in addition to carotenoids, produces such compounds as chlorophyll and tocopherols, Vitamin E active agents. The carotenoid pathway in plants produces carotenes, such as xcex1- and xcex2carotene, and lycopene, and xanthophylls, such as lutein.
The biosynthesis of carotenoids involves the condensation of two molecules of the C20 precursor geranyl PPi to yield the first C40 hydrocarbon phytoene. In a series of sequential desaturatioris, phytoene yields lycopene. Lycopene is the precursor of the cyclic carotenes, xcex2-carotene and a xcex1-carotene. The xanthophylls, zeaxanthin and lutein are formed by hydroxylation of xcex2-carotene and a xcex1-carotene, respectively.
xcex2carotene, a carotene whose color is in the spectrum ranging from yellow to orange, is present in a large amount in the roots of carrots and in green leaves of plants. xcex2carotene is useful as a coloring material and also as a precursor of vitamin A in mammals. Current methods for commercial production of xcex2-carotene include isolation from carrots, chemical synthesis, and microbial production.
A number of crop plants and a single oilseed crop are known to have substantial levels of carotenoids, and consumption of such natural sources of carotenoids have been indicated as providing various beneficial health effects. The below table provides levels of carotenoids that have been reported for various plant species.
The pathway for biosynthesis of the carotenoids has been studied in a variety of organisms and the biosynthetic pathway has been elucidated in organisms ranging from bacteria to higher plants. See, for example, Britton, G. (1988) Biosynthesis of carotenoids, p. 133-182, In T. W. Goodwin (ed.), Plant pigments, 1988. Academic Press, Inc. (London), Ltd., London. Carotenoid biosynthesis genes have also been cloned from a variety of organisms including Erwinia uredovora (Misawa et al. (1990) J. Bacteriol. 172:6704-6712; Erwinia herbicola (Application WO 91/13078, Armstrong et al. (1990) Proc. Nat. Acad. Sci., USA 87:9975-9979); R. capsulatus (Armstrong et al. (1989) Mol. Gen. Genet. 216:254-268, Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421); Thermus thermophilus (Hoshino et al. (1993) Appl. Environ. Microbiol. 59:3150-3153); the cyanobacterium Synechococcus sp. (Genbank accession number X63873). See also, application WO 96/13149 and the references cited therein.
While the genes have been elucidated, little is known about the use of the genes in plants. Investigations have shown that over expression or inhibition of expression of the plant phytoene synthase (Psy1) gene in transgenic plants can alter carotenoid levels in fruits. See, Bird et al. (1991) Biotechnology 9:635-639; Bramley et al. (1992) Plant J. 2:343-349; and Fray and Grierson (1993) Plant Mol. Biol. 22:589-602. Further, as reported by Fray et al. (1995) The Plant Journal 8:693-701, constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway.
Application WO 96/13149 reports on enhancing carotenoid accumulation in storage organs such as tubers and roots of genetically engineered plants. The application is directed towards enhancing colored native carotenoid production in specific, predetermined non-photosynthetic storage organs. The examples of the application are drawn to increasing colored carotenoids in transformed carrot roots and in orange flesh potato tubers. Both of these tissues are vegetative tissues, not seeds, and natively have a high level of carotenoids.
Carotenoids are useful in a variety of applications. Generally, carotenoids are useful as supplements, particularly vitamin supplements, as vegetable oil based food products and food ingredents, as feed additives in animal feeds and as colorants. Specifically, phytoene finds use in treating skin disorders. See, for example, U.S. Pat. No. 4,642,318. Lycopene, xcex1- and xcex2-carotene are used as food coloring agents. Consumption of xcex2-carotene and lycopene has also been implicated as having preventative effects against certain kinds of cancers. In addition, lutein consumption has been associated with prevention of macular degeneration of the eye.
Plant oils are useful in a variety of industrial and edible applications. Novel vegetable oils compositions and/or improved means to obtain oils compositions, from biosynthetic or natural plant sources are needed. Depending upon the intended oil use, various different fatty acid compositions are desired. The demand for modified oils with specific fatty acid compositions is great, particularly for oils high in oleic acid. See, Haumann, B. F. (1996) INFORM 7:320-334. As reported by Haumann, the ideal frying oil would be a low-saturate, high oleic and low linolenic oil. Furthermore, studies in recent years have established the value of monounsaturated fatty acids as a dietary constituent.
Attempts have been made over the years to improve the fatty acid profiles of particular oils. For example, the oxidative stability of vegetable oil is related to the number of double bonds in its fatty acids. That is, molecules with several double bonds are recognized to be more unstable. Thus, scientists have attempted to reduce the content of xcex1-linolenic acid in order to improve shelf life and oxidative stability, particularly under heat.
It is apparent that there is needed a method for producing significant levels of carotenoid compounds in crop plants and particularly in plant seeds. It would additionally be beneficial to alter the fatty acid content of the plants and seeds. Such altered seed products would be useful nutritionally as well as provide a source for producing more stable oils. There is no report of methods to substantially altering the levels and composition of carotenoids produced in a plant seed, particularly with respect to increasing the level of production of carotenoids. There is therefore needed, a useful method for altering carotenoid levels in plants, particularly seeds, and for producing oils with modified carotenoid composition and/or content.
A number of unique and interconnected biochemical pathways leading to secondary metabolites, including tocopherols, exist in chloroplasts of higher plants. Tocopherols not only perform vital functions in plants, but are also important from mammalian nutritional perspectives. In plastids, tocopherols account for up to 40% of the total quinone pool. As shown in FIG. 15, the biosynthesis of xcex1-tocopherol in higher plants involves condensation of homogentisic acid and phytylpyrophosphate to form 2-methyl-6 phytylbenzoquinol that can, by cyclization and subsequent methylations (Fiedler et al., 1982, Planta, 155: 511-515, Soll et al., 1980, Arch. Biochem. Biophys. 204: 544-550, Marshall et al., 1985 Phytochem., 24: 1705-1711, all of which are herein incorporated by reference in their entirety), form various tocopherols. Considering the structure and source from which the structural moieties can be derived, the plant tocopherol biosynthetic pathway can be divided into four parts: formation of homogentisic acid; synthesis of phytylpyrophosphate; cyclization; and S-adenosylmethionine-dependent methylation of the aromatic ring.
PCT International Application WO 97/27285 discloses a cDNA clone from Arabidopsis thaliana that encodes p-hydroxyphenyl pyruvic acid dioxygenase (OHPP dioxygenase, or HPD), which catalyzes the production of homogentisic acid from the shikimate pathway intermediate p-hydroxyphenyl pyruvic acid via an oxidation/decarboxylation reaction. This application also discloses a method of creating a transgenic plant in which the levels of OHPP dioxygenase are elevated sufficiently such that production of plastoquinones, Vitamin E, and carotenoids are modified. Organelle targetting of the OHPP dioxygenase is not discussed.
Tocopherols and tocotrienols (unsaturated tocopherol derivatives) are well known antioxidants, and play an important role in protecting cells from free radical damage, and in the prevention of many diseases, including cardiac disease, cancer, cataracts, retinopathy, Alzheimer""s disease, and neurodegeneration, and have been shown to have beneficial effects on symptoms of arthritis, and in anti-aging. Vitamin E is used in chicken feed for improving the shelf life, appearance, flavor, and oxidative stability of meat, and to transfer tocols from feed to eggs. Vitamin E has been shown to be essential for normal reproduction, improves overall performance, and enhances immunocompetence in livestock animals. Vitamin E supplement in animal feed also imparts oxidative stability to milk products.
The demand for natural tocopherols as supplements has been steadily growing at a rate of 10-20% for the past three years. At present, the demand exceeds the supply for natural tocopherols, which are known to be more biopotent than racemic mixtures of synthetically produced tocopherols. Naturally occurring tocopherols are all d-stereomers, whereas synthetic xcex1-tocopherol is a mixture of eight d,l-xcex1-tocopherol isomers, only one of which (12.5%) is identical to the natural d-xcex1-tocopherol. Natural d-xcex1-tocopherol has the highest vitamin E activity (1.49 IU/mg) when compared to other natural topherols or tocotrienols. The synthetic xcex1-tocopherol has a vitamin E activity of 1.1 IU/mg. In 1995, the worldwide market for raw refined tocopherols was $1020 million; synthetic materials comprised 85-88% of the market, the remaining 12-15% being natural materials. The best sources of natural tocopherols and tocotrienols are vegetable oils and grain products. Currently, most of the natural Vitamin E is produced from xcex3-tocopherol derived from soy oil processing, which is subsequently converted to xcex1-tocopherol by chemical modification (xcex1-tocopherol exhibits the greatest biological activity).
Methods of enhancing the levels of tocopherols and tocotrienols in oil seeds and cereal grains, especially levels of the more desirable compounds that can be used directly, without chemical modification, would be useful to the art as such molecules exhibit better functionality and biovailability.
Transformed plants, plant cells and seeds having altered carotenoid levels and/or modified fatty acid compositions, as well as altered tocopherol levels and composition, are provided. The plants, plant cells and seeds are transformed with at least one carotenoid biosynthesis gene, one tocopherol biosynthesis gene, or a combination thereof. Methods for making and using the transformed compositions of the invention are also provided. Methods find use in altering carotenoid and tocopherol levels in plants, particularly seeds, as well as increasing particular compounds for molecular farming, such as for production of particular carotenoids and tocopherols. At the same time, the transformed compositions, particularly seeds, provide a source of modified oils, which oils may be extracted from the seeds in order to provide an oil product comprising a natural source of various carotenoids, carotenoid mixtures, individual tocopherol compounds, and tocopherol mixtures. In a particular aspect of the present invention, transformed seed can provide a source for particular carotenoid compounds and/or for modified speciality oils having altered carotenoid or tocopherol compositions and/or altered fatty acid composition, particularly having increased levels of oleic acid and decreased levels of linoleic and linolenic acids, and increased levels of xcex1-tocopherol.